STRUCTURE, FUNCTION, & DYNAMICS OF P-450 CYTOCHROMES

结构、功能、

• 批准号: 3466227
• 负责人: SHAUN BLACK
• 金额: $ 8.11万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466227
• 关键词: NADPH cytochrome c2 reductase; X ray crystallography; affinity chromatography; chemical structure function; circular dichroism; computer data analysis; conformation; cytochrome P450; cytochrome b5 reductase; enzyme linked immunosorbent assay; enzyme model; enzyme structure; hemoprotein structure; high performance liquid chromatography; isozymes; laboratory rabbit; membrane proteins; membrane structure; microsomes; molecular site; monoclonal antibody; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein sequence; synthetic peptide

Structure, function, and conformational dynamics of the cytochrome P-450 monooxygenase system are to be studied in the proposed research. (1) Topology of membrane-bound rabbit microsomal isozymes 2 and 4 will be probed via polyclonal and monoclonal antibodies as well as by specific and non-specific proteolysis. Immobilized polyclonal antibodies will be used to purify specific peptides released upon protease treatment of microsomes; HPLC-isolated peptides will be submitted to micro- sequence analysis to reveal sites within the primary structures available at the external (cytoplasmic) surface of the endoplasmic reticulum. A panel of monoclonal antibodies will be raised to synthetic peptides selected from each sequence by MSEQ computer analysis; these site-specific antibodies will be utilized to probe native and protease-digested microsomes via ELISA and Western-blotting procedures. Together, the results obtained will permit reconstruction of the membrane topological features of each cytochrome. Preliminary data and calculations suggest, in contrast to current theory, that the topology of the P-450's is simple and is similar to that known for other microsomal proteins. Isozymes 2 and 4 were chosen for the proposed studies as they are the major forms present in phenobarbital-induced (isozyme 2) and aryl hydrocarbon-induced (isozyme 4) animals, they represent two major gene sub-families, they typify low- and high-spin cytochrome types, they are involved in drug disposition tolerance, and they participate in the oxidative metabolism of physiological lipids, numerous drugs, and other xenobiotics; furthermore, they are highly similar to forms known in the human. (2) Conformational dynamics of isozyme 2 will be studied with site- specific chemical modification of Cys-152, a residue shown to serve as a "reporter" for binding events at the active site. Modification kinetics, carbon-13 NMR spectroscopy, and circular dichroism will be utilized to examine the role and importance of conformational changes in the function of the cytochrome. Functional aspects to be examined will include substrate binding, heme reduction kinetics, and protein-protein interactions. (3) Crystallization of many of the microsomal monooxygenase system components will be attempted with the ultimate aim of three- dimensional structure elucidation. Exploration of the "small Amphiphile" concept and co-crystallization will be major thrusts. A long-term objective of these studies is to achieve a sophisticated molecular view of the P-450 system that will serve to guide rational drug design.

的结构、功能和构象动力学 细胞色素P-450单加氧酶系统将在 提议的研究。 (1)膜束缚兔的拓扑学 微粒体同工酶2和4将通过多克隆和 单克隆抗体以及通过特异性和非特异性 蛋白水解 固定化多克隆抗体将用于 纯化蛋白酶处理后释放的特异性肽， 微粒体; HPLC分离的肽将提交给微粒体， 序列分析以揭示一级结构内的位点 在内质网的外（细胞质）表面 网状细胞。 一组单克隆抗体将被提高到 通过MSEQ从每个序列中选择的合成肽 计算机分析;将利用这些位点特异性抗体 通过ELISA探测天然和蛋白酶消化的微粒体， 蛋白质印迹程序。 总之，所取得的成果将 允许重构膜的拓扑特征， 每个细胞色素 初步数据和计算表明， 与目前的理论相反，P-450的拓扑结构是 简单，类似于已知的其他微粒体蛋白质。 选择同工酶2和4用于拟定的研究，因为它们是 苯巴比妥诱导的（同工酶2）和 芳烃诱导的（同工酶4）动物，它们代表两种 主要的基因亚家族，他们代表低和高自旋 细胞色素类型，它们参与药物处置耐受性， 它们参与生理性氧化代谢， 脂质，许多药物和其他外源性物质;此外，它们 与人类已知的形式高度相似。 （二） 同工酶2的构象动力学将用位点- Cys-152的特异性化学修饰， 作为活性位点结合事件的“报告者”。 改性动力学，碳-13 NMR光谱，和循环 二向色性将被用来研究的作用和重要性， 细胞色素功能的构象变化。 要检查的功能方面将包括底物结合， 血红素还原动力学和蛋白质-蛋白质相互作用。 （三） 许多微粒体单加氧酶系统的结晶 我们将努力实现以下三个目标： 空间结构解析 探索“小 “两亲”概念和共结晶将是主要的突破口。 这些研究的一个长期目标是实现 P-450系统的复杂分子视图， 指导合理的药物设计。